MXPA00007796A - A process for the fermentative preparation of metabolic products and for the nucleotide sequences encoding sod. - Google Patents

Abstract

The invention provides nucleotide sequences encoding for the sod gene and a process for the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine, using coryneform bacteria in which the sod gene is amplified.

Description

PROCESS FOR THE FERMENTATIVE PREPARATION OF METABOLIC PRODUCTS AND FOR THE NUCLEOTIDE SEQUENCES THAT CODIFY FOR THE SOD GENE FIELD OF THE INVENTION The invention provides the nucleotide sequences coding for the sod gene and a process for the fermentative preparation of nucleotides, vitamins and L-a inocides, in particular L-lysine using coryneform bacteria in which the sod gene is amplified.

PREVIOUS TECHNIQUE Nucleotides, vitamins and L-amino acids, in particular L-lysine, are used in the food industry, in animal nutrition, in human medicine and in the pharmaceutical industry. It is known that these substances can be prepared by fermenting strains of coryneform bacteria, in particular Coryn eba c t eri um gl u t ami cum. Constant efforts are made to improve the preparation method REF: 122046 due to the high degree of importance of these substances. The improvements in the process can be related to the engineering factors of the fermentation such as, for example, agitation and oxygen supply, or the composition of the nutrient medium, such as, for example, the concentration of the sugar during fermentation, or the treatment process. aimed at obtaining the product itself, for example by ion exchange chromatography or the intrinsic potency of the microorganism itself. To improve the potency of the microorganisms, the methods of mutagenesis, selection and choice of mutants are used. Strains that are resistant to antimetabolites or which are auxotrophic for significant regulatory intermediates, are obtained in this way and produce nucleotides, vitamins and amino acids. For some time, now recombinant DNA engineering methods have also been used for the improvement of strains, producing strains of L-amino acids of Coryn eba ct eri um gl u tami cum, by amplifying the biosynthetic amino acid genes, individual, and investigating the effect on the production of La inoácido. U.S. Patent No. 5,179,010 describes strains of coryneform bacteria which are for example resistant to ethylviologen or benzoyl peroxide, have an increased activity to superoxide dismutase and have an improved lysine yield. These strains were produced by non-directed mutagenesis using the mutagen N-raethyl-N-nitro-N-nitrosoguanidine. The increase in the concentration of superoxide-disutase in the strains mentioned there was at most 56%. U.S. Patent No. 4,529,697 describes mutants of coryneform bacteria that produce glutamic acid. The increase in the concentration of superoxide dismutase in the strains mentioned there was at most 105%.

OBJECTIVE OF THE INVENTION The inventor has formulated the objective as the provision of new steps for the improved handling of superoxide dismutase from coryneform bacteria. These steps can be used during the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine.

DESCRIPTION OF THE INVENTION Nucleotides, vitamins and L-amino acids, in particular L-lysine, are used in the food industry, in animal nutrition, in human medicine and in the pharmaceutical industry. The lysine-producing strains of coryneform bacteria are known from the prior art, in which the concentration of superoxide dismutase is increased by 27 to 56% and which release amplified lysine. These strains were obtained by h or directed mutagenesis. Whenever L-lysine or lysine is mentioned in the following, it is intended that this not only mean the base but also salts such as, for example, lysine monohydrochloride or lysine sulfate. The invention provides a preferably recombinant DNA from Coryn eba ct erium source which can be replicated in coryneform microorganisms and which contains at least the nucleotide sequence coding for the sod gene represented in SEQ ID NO. The invention also provides a replicable DNA according to Claim 1 comprising: i) the nucleotide sequence, shown in SEQ ID NO. 1, or ii) at least one sequence corresponding to the sequence (i) within the region of degeneracy of the genetic code, or iii) at least one sequence that hybridizes with the sequence that is complementary to the sequence (i) or (ii) and optionally iv) sense, functionally neutral mutations in (i). Coryneform microorganisms, in particular the strain Coryn eba ct erium, transformed by the introduction of the aforementioned replicable DNA, are also provided by the present invention. In addition, the invention provides a process for the fermentative preparation of nucleotides, vitamins and L-amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce the relevant product and in which the nucleotide sequences coding for the sod gene they are amplified, in particular they are overexpressed. The term "amplification" in this context describes the increase in the intracellular activity of one or more enzymes in a microorganism, which are encoded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a strong promoter. or a gene encoding a corresponding enzyme with high activity, and optionally combining these steps. The microorganisms which are the subject of the present invention can produce nucleotides, vitamins and L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol . These can be members of the family of coryneform bacteria in particular of the genus Coryneba c t eri um. In the case of gender Coryn eba c t eri um, in particular the species Coryn eba c t eri um gl u tami cum, I should mention that it is well known in the specialized field for its ability to produce L-amino acids.

Strains suitable for the genus Corynebacterium, in particular the species Corynebacterium glutamicum are for example the wildtype strains known Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium ammoniagenes ATCC6871 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lacto ermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 Corynebacterium melassecola ATCC17965 Brevibacterium ammoniagenes IF012072 and mutants or strains prepared therefrom, which can produce nucleotides, vitamins and L-amino acids, such as for example the 5'-inosinic acid strains Corynebacterium ammoniagenes ATCC15190 Corynebacterium ammoniagenes ATCC15454 Corynebacterium glutamicum ATCC14998 or such as for example the acid producing strains 5'-guanylic Corynebacterium glutamicum ATCC21171 or Corynebacterium ammoniagenes ATCC19216 or such as for example the producers of L-lysine Corynebac terium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P-1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 and Corynebacterium glutamicum FERM-P 6464 Corynebacterium glutamicum DSM 5714.

The inventors were able to isolate the new sod gene from Corynebacterium melassecola ATCC17965. Here, the enzyme protein superoxide dismutase was first purified to homogeneity using chromatographic methods. The methods and instructions for the purification and preparation of proteins are completely described for example in the textbook by Schleifer and Wensink: Practical Methods in Molecular Biology (Springer Verlag, Berlin, Germany, 1981), in the manual by Harris and Angal: Protein Purification Methods: A Practical approach (IRL Press, Oxford, United Kingdom, 1989), in the textbook by Scopes: Protein Purification: Principles and Practice, 3rd ed. (Springer Verlag, New York, USA, 1993) and in general in well-known textbooks and manuals. The pure enzymatic protein can then be cleaved into peptides by treatment with suitable enzymes such as for example trypsin or chymotrypsin. The amino acid sequence in these peptides can be determined by the N-terminal sequencing method described by Edman (Archives of Biochemistry 22, 475, (1949)). Methods and instructions for protein sequencing are given for example in Smith: Protein Sequencing Protocols: Methods in Molecular Biology, Vol. 64 and Vol. 112 (Humana Press, Toto a, NJ, USA, 1996) and in Ka p et al .: Protein Structure Analysis: Preparation, Characterization and Microsequencing (Springer Verlag, New York, NY, USA, 1997). The amino acid sequence in the enzyme protein superoxide dismutase can be partially or completely determined in this way, depending on the degree of complexity. By exploiting the known use of a codon for coryneform bacteria (Malumbres et al. (Gene 134, 15-24 (1993)), synthetic oligonucleotides can be synthesized and used as primers to amplify the corresponding chromosomal DNA segments by means of the polymerase chain reaction (PCR) The instructions for this can be found by a person skilled in the art, among others, for example in the manual by Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, United Kingdom, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) The DNA fragment of the sod gene obtained in this way is then cloned using known methods such as described for example in Sambrook et al. : Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press, USA, 1989) and can be used as probes to test the complete gene, including its 5 'and 3' flanks in gene banks. The construction of gene banks is generally described in well-known textbooks and manuals in general. The following can be mentioned as examples, the textbook by Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al .: Molecular Cloning, A Laboratory Manual ( Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is that of E. coli K-12 strain W3110 that was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in vectors? Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank of C. gl u tami cum ATCC13032, which was constructed with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Bormann et al. (Molecular Microbiology 6 (3), 317-326)) also describe a gene bank of C. gl u tami cum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-198 (1980)). To produce a gene bank of C. gl ut ami cum in E. coli, plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can also be used. Suitable hosts are in particular those strains of E. coli that are defective in restriction and recombination. An example of these is the strain DH5amcr described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87, (1990) 4645-4649). The long DNA fragments cloned with the help of cosmids can then be subcloned back into vectors currently used for sequencing, and then sequenced as described in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America USA, - 74: 5463-5467, 1977). The DNA sequences obtained can then be tested with known algorithms or sequence analysis programs such as that of. Staden (Nucleic Acids Research 14, 217-232 (1986)), the GCG program by Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) the algorithm FASTA by Pearson 'and Lipman (Proceedings of the National Academy of Sciences' USA 85, 2444-2448 (1988)) or the BLAST algorithm of Altschul et al.

(Nature Genetics 6, 119-129 (1994)) and compared to the sequence records present in publicly accessible data banks. Publicly accessible libraries for nucleotide sequences are for example those at European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or those at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).

The new DNA sequences of C. gl u tami cum coding for the sod gene, which can be obtained in this manner, are a constituent of the present invention as SEQ ID NO. 1. In addition, the amino sequence of the corresponding protein was derived from the DNA sequence present. The amino acid sequence of the sod gene product is represented in SEQ ID NO. 2. The coding DNA sequence, produced from SEQ ID NO. 1 for the degeneracy of the genetic code, is also a constituent of the invention. In the same way, the DNA sequences that hybridize with SEQ ID NO. 1 or parts of SEQ ID NO. 1 are also a constituent of the invention. In the specialized field, moreover, conservative amino acid exchanges such as for example the exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, as "mutations in sense", are also known, and do not lead to any basic modification in the activity of the protein, for example these are functionally neutral. In addition, it is known that changes to the N-terminus and / or the C-terminus of a protein can not substantially impair or even stabilize its function. The data related to these can be found by a person skilled in the art, among others, in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), in O'Regan et al. (Gene 77: 237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)), in Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in well-known textbooks on genetics and molecular biology. The amino acid sequences that are produced in a corresponding manner from SEQ ID NO. 2 are also a constituent of the invention. The amplification of the sod gene in coryneform bacteria leads to an unusually high increase in the concentration of superoxide dismutase in the microorganism. To produce an overexpression, the number of copies of the corresponding gene can be increased, or the promoter and regulatory region or the ribosome binding site, which is located upstream of the structure gene, can be mutated. The expression cassettes, which are incorporated upstream (5 ') of the structural gene, operate in the same way. It is also possible to increase the expression during the course of the fermentative production of L-lysine with inducible promoters. The expression is also improved by measures aimed at prolonging the lifetime of the mRNA. In addition, the enzymatic activity can also be amplified by inhibiting the degradation of the enzyme protein. Genes or gene constructs can either be present in plasmids with different copy numbers or be integrated and amplified in the chromosome. Alternatively, overexpression of the relevant genes can also be achieved by modifying the composition of the media and the management of the culture. The instructions for these procedures can be found by a person skilled in the art, among others, in Martin et al.

(Bio / Technology 5, 137-146 (1987)), in Guerrero et al (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-99 (1991)), in European patent EP-B-0, 472, 869, in the United States Patent No. 4,601,893, in Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in Labarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application W096 / 15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in Japanese patent document JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering, 58, 191-195 (1998)), in Makrides (Microbiological Reviews, 60: 512-538 (1996)) and in well-known textbooks on genetics and molecular biology. An example of a plasmid with the help of which the sod gene can be overexpressed is pMM23 (Figure 1) which is contained in strain MH20-22B / pMM23. Plasmid pMM23 is a shuttle vector of E. coli-C. glutamicum based on plasmid pBL1 (Fernandes-Gonzales et al., Journal of Bacteriology 176 (11), 3154-3161 (1994)), pACYC184 (Chan and Cohen, Journal of Bacteriology 134 (3), 1141-1156 (1978)) and the tre promoter (Brosius et al., Journal of Biological Chemistry 260, 3539-3541 (1985)) which possesses the sod gene. Other plasmid vectors that can be replicated in C. gl u tami cum such as for example pEKExl (Eikmanns et al., Gene 102: 93-98 (1991)) or pZ8-l (European Patent 0,375,889) can be used the same way that the initial vectors to clone and express the sod gene. This may also be advantageous for the production of nucleotides, vitamins and in particular L-amino acids, to overexpress one or more enzymes in the particular biosynthetic pathway, in addition to the sod gene. Thus, for example, for the preparation of nucleotides "the purF gene encoding the glutamine PRPP-amidotransferase can be simultaneously overexpressed." The carAB gene encoding the carbamoyl phosphate synthetase can be simultaneously overexpressed.

Thus, for example, for the preparation of D-pantothenic acid "the panD gene coding for aspartate-decarboxylase (Dusch et al., Applied and Environmental Microbiology 65, 1530-1539 (1999)) can be simultaneously overexpressed.

Finally, for example, for the preparation of L-lysine m the dapA gene coding for dihydrodipicolinate synthase can be simultaneously overexpressed (EP-B-0.197.335), or "a DNA fragment that promotes resistance to S- ( 2-aminoethyl) -cysteine can be simultaneously amplified (EP-A-0, 088, 166).

Furthermore, it may be advantageous for the production of nucleotides, vitamins and in particular L-amino acids, very particularly L-lysine, to change or displace unwanted side reactions apart from the overexpression of the sod gene (Nakayama: "Breeding of Amino Acid"). Producing Micro-organisms ", in Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.) Academic Press, London, United Kingdom, 1982). The microorganisms prepared according to the invention can be grown continuously or in batches in a batch process or in a batch feeding process, or in a batch fed repeated process for the purpose of producing the metabolic products. A review of the known cultivation methods is given in the textbook by Chiel (Bioprozess technik 1. Einfuhrung in die Bioverfahrens technik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)). The culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the book "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981). Sugar and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as for example soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as for example palmitic acid, stearic acid and linoleic acid, alcohols such as for example glycerol and ethanol and organic acids such such as acetic acid can be used as carbon sources. These substances can be used individually or as a mixture. Nitrogen-containing organic compounds such as peptones, yeast extract, meat extract, malt extract, corn extraction water, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium carbonate and ammonium nitrate can be used as nitrogen sources. The nitrogen sources can be used individually or as a mixture. The phosphoric acids, the potassium diacid phosphate or the dipotassium acid phosphate or the corresponding salts containing sodium, can be used as sources of phosphorus. The culture medium should also contain metal salts such as for example magnesium sulfate or iron sulfate, which are required for growth. Finally, essential growth substances such as amino acids and vitamins, in addition to the substances mentioned above, can also be used. Above and above these, suitable precursors may also be added to the culture medium. The aforementioned feedstocks can be added to the culture as a single batch or can be supplied during cultivation in an appropriate manner. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid, may be used in the proper manner to control the pH of the culture. Antifoam agents such as for example polyglycol fatty acid esters can be used to control the production of the foam. To maintain the stability of the plasmids, suitable selective action substances, for example antibiotics, can be added to the medium. In order to maintain aerobic conditions, oxygen gas or oxygen-containing mixtures, such as for example air, as well as oxygen-containing compounds, such as, for example, peroxide compounds, in particular peroxide, may be introduced into the culture. of hydrogen. The culture temperature is generally 20 ° C to 45 ° C and preferably 25 ° C to 40 ° C. The cultivation is continued until a maximum in the desired L-amino acid has been produced. This goal is normally achieved within 10 hours to 160 hours.

The following microorganisms were deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) according to the Budapest treaty: "Escherichia coli strain XLl blue / pMM23 as DSM 12860"Corynebacterium melassecol to strain 1019 and DSM 12859.

The process according to the invention is used for the fermentative preparation of nucleotides, vitamins and in particular L-amino acids with "coryneform bacteria, very particularly the preparation of L-lysine." The following figures are attached: "Figure 1: Diagram of the plasmid pMM23"Figure 2: Diagram of plasmid pCGL482.

The values for the data related to the numbers of base pairs obtained are within the scope of reproducibility. The abbreviations used are explained below: Ptrc: Promoter tre Sod: Superoxide dismutase gene cat: Chloramphenicol resistance gene ori pBLl: Replication region of plasmid pBLl ori pACYC Replication region of plasmid pACYC184 ori M13: Replication region of phage M13 p: Base pair Notl: restriction endonuclease cleavage site Notl Xbal: restriction endonuclease cleavage site Xbal BamHI: restriction endonuclease site BamHI Ncol restriction endonuclease site Ncol exit endonuclease site of restriction Salí Bglll Cutting point of the restriction endonuclease BglII Ndel: Cut-off point of the restriction endonuclease Ndel Smal: Cut-off point of the restriction endonuclease Smal PstI: Cut-off point of the restriction endonuclease PstI Stul Cut-off point of restriction endonuclease Stul Xhol Cut-off point of restriction endonuclease Xhol EXAMPLES The following examples will further illustrate this invention.

Example 1 Cloning and Sequencing of the sod gene 1. Purification and Characterization of the superoxide dismutase of Corynebacterium mel assecol ATCC17965 For the purification of superoxide dismutase, Coryn eba ct eri um mel was developed at ATCC17965 aerobically on Bacto® Brain-Heart Infusion Medium (DIFCO Laboratories, Detroit, USA) at 34 ° C until the late exponential phase. Cells from a 1 liter culture were harvested by centrifugation using an Avantió J-25 centrifuge, and a JLA-10,500 rotor, from BECKMANN (Palo Alto, USA) at 5,000 xg for 15 minutes at 4 ° C, and resuspended in a solution of 10 mM MgCl 2, 1 μg / ml of Ribonuclease A, 1 μg / ml of deoxyribonuclease I, 25 mM of sodium phosphate buffer pH 7.5, to give a final optical density of 500, as measured by a spectrophotometer of BECKMAN (Palo Alto, USA) at a wavelength of 570 nm. For disintegration, the cells were passed three times through a Manual-Fill 40K cell (FA-030) with a FRENCH® pressurized cell press from SLM AMINCO® (Urbana, USA) at a cutting force of 110 'MPa (16,000 psi). The resulting extract was centrifuged using an Avantid J-25 centrifuge and a JA-25.50 rotor from BECKMAN (Palo Alto, USA) at 10,000 x g for 15 minutes at 4 ° C. The supernatant was ultracentrifuged using an L8-70M ultracentrifuge and a VTi 65.2 rotor from BECKMA-N (Palo Alto, USA) at 130,000 per g for 2 hours at 4 ° C. The supernatant was used as the crude extract. From this step, the purification of superoxide dismutase was followed by activity staining after non-denaturing gel electrophoresis, as described by Schmidt et al (European Journal of Biochemistry 156, 149-155 (1986)) or by specific enzymatic assay described by Ukeda et al. (Analytical Biochemistry 251 (2), 206-209 (1997)), and by analysis of • SDS-PAGE as described by Laemmli (Nature 227, 680-685 (1970)). The protein content was estimated by the Lowry method (Lowry et al., Journal of Biolagical Chemistry 193, 265-275 (1951)) using Bovine Immunoglobulin G as a standard. The crude extract was precipitated for 30 minutes on ice by the addition of crystalline ammonium sulfate to a final saturation of 90%. The precipitate was collected by centrifugation at 10 ° C., 000 x g for 15 minutes at 4 ° C, using an Avantió J-25 centrifuge and a JA-25.50 rotor from BECKMAN (Palo Alto, USA), and discarded. The supernatant was adjusted to a saturation of 100% ammonium sulfate and the precipitate was collected by centrifugation as described above. The button or concentrate was dissolved in 10 ml of 50 mM Tris-Cl buffer, pH 8.0. This solution was dialysed for 24 hours against 2 liters of the same buffer. The protein solution was applied to a 2 ml Bioscale Q2 ion exchange column of BIO-RAD (Hercules, USA), the column was washed with two column volumes of 50 M Tris-Cl buffer, pH 8.0, and then it was eluted with a linear gradient of sodium chloride 0-0.5 M. The active fractions were combined, dialysed against the buffer of 50 mM Tris-Cl, pH 8.0. Proteins were separated by gel filtration on a 100 ml Sephacryl S300 column of PHARMACIA Biotech (Saint Quentin in Yvelines, France). All the chromatographic steps were carried out with the FPLC BioLogic HR system from BIO-RAD (Hercules, USA). The active fractions were combined and concentrated with Centricon®-30 concentrator cartridges from AMICON (Epernon, France). The final solution of superoxide dismutase had a volume of 540 μl with a protein concentration of 0.2 mg / ml as estimated by the Lowry method (Lowry et al., Journal of Biological Chemistry 193, 265-275 (1951)) . It was estimated that the superdioxide dismutase was pure, by SDS-PAGE analysis (Laemmli, Nature 227, 680-685 (1970)), and its subunit molecular weight was estimated as 24.5 kDa.

Pure superoxide dismutase (10 μg) was isolated from an acrylamide gel of SDS-PAGE within a slice. The protein was digested in the gel slice with 0.2 μg of trypsin in 200 μl of 0.01% TWEEN 20, 0.1 M Tris-Cl buffer, pH 8.6. The tryptic peptides were separated by a linear gradient in acetonitrile / 0.1% trifluoroacetic acid, on a DEAE-C18 column with a HPLC chromatography system 104A from APPLIED BIOSYSTEMS (Foster City, USA). Two isolated peptides were sequenced with an APPLIED BIOSYSTEMS 473A Sequencer (Foster City, USA). These peptides have the following sequence: Peptide 15: NLAFNLGGHTNHSVF Peptide 18: FQDHFNSAALGLQGS A comparison, using the BLAST program (Altschul et al., Journal of Molecular Biology 215, 403-410 (1990)), of the peptides sequenced with the sequences of protein in the database of the National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov) revealed high identities with the superoxide dismutases of Actinomyces vi s cos us (100% identity, accession number X81381), Corynebacteri um diph teri ae (86% of identity, accession number X81382) for peptide 15, and Coryneba cteri um diph teri ae (78% identity, accession number X81382) and Coryn eba ct erium pse udodiph t eri ti cum (73% identity, number access X81383) for peptide 18. 2. Cloning of the sod gene The molecular biology techniques used in the cloning experiments were described by Ausubel et al. (Ausubel et al., In: Cúrrente protocols in molecular biology, John Wiley and sons (eds.), New York, USA (1987)), except when it was established otherwise. Molecular biology products were purchased from PROMEGA CORPORATION (Madison, USA) or from BOEHRINGER MANNHEIM FRANCE S.A. (Meylan, France), except 'when specified. To clone the sod gene coding for superoxide dismutase, a bank of genomic DNA was constructed. Corynejbacterium mel a sesecol was developed at ATCC17965 aerobically on Brain Heart Infusion Medium (DIFCO Laboratories, Detroit, USA) at 34 ° C until the late exponential phase. Cells from a 25 ml culture were harvested by centrifugation using an Avantio J-25 centrifuge, and a JLA-10,500 rotor, from BECKMANN (Palo Alto, USA) at 5,000 xg for 15 minutes at 4 ° C, resuspended in 3 ml of 10 mM ethylenediaminetetraacetate, 1% glucose, 20 mg / ml lysozyme, 25 mM Tris-Cl buffer pH 8.5, and incubated at 37 ° C for 1 hour. Cell lysis was achieved by the addition of 6.5 ml of 1 mM ethylenediaminetetraacetate, 0.5% sodium dodecyl sulfate, 1 mg / ml proteinase K, 10 mM Tris-Cl buffer pH 7.5, and incubation at 37 ° C for 1 hour. To this suspension, 1.8 ml of 5 M NaCl and 1.5 ml of 10% cetyltridecylammonium bromide, 0.7 M sodium chloride solution were added. After incubation at 60 ° C for 20 minutes, the chromosomal DNA was extracted by gentle mixture with an equal volume of chloroform / isoamyl alcohol solution 24: 1 V / V. The chromosomal DNA in the aqueous phase was precipitated with 0.6 volumes of isopropanol. The DNA precipitate was recovered by centrifugation at 10,000 x g for 15 minutes at 4 ° C, using an Avantió J-25 centrifuge and a JA-25.50 rotor from BECKMAN (Palo Alto, USA), and resuspended in 1 mM EDTA, 10 mM Tris-Cl buffer pH 7.5. To 1 mg of the chromosomal DNA diluted in the appropriate buffer, 15 units of the restriction enzyme Mbo I were added, and the mixture was incubated for 1 hour at 37 ° C. Partially restricted DNA was fractionated by centrifugation in a gradient of sucrose at 10-40% P / V, using an ultracentrifuge L8-70M and a SW40TÍ rotor from BECKMAN (Palo Alto, USA) at 80,000 xg for 17 hours at 20 ° C. Chromosomal AUN fragments in the size range of 6 to 10 kb, as estimated by horizontal agarose gel electrophoresis, were recovered and combined. The pUN121 cloning vector positive for selection (Nilsson et al., Nucleic Acids Research 11, 8019-8030 (1983)) was linearized by the restriction enzyme Bel I. At 1 μg of linearized pUN 121, 2 μg of Chromosomal DNA fragments generated by Mbol, from Coryn eba ct erium mel assecola ATCC17965, in a ligation mixture containing 0.1 unit of T4 DNA ligase, incubated for 24 hours at 16 ° C. The ligation mixture was then used to transform the DH5a strain of Es ch eri qui a col i (Hanahan, Journal of Molecular Biology 166, 557-580 (1983)) by electroporation as described by Bonamy et al.

(Bonamy et al., FEMS Microbiology Letters 66, 263-270 (1990)). The transformants were recovered after incubation on 10 μg / ml Luria Bertani agar from Bacto®, which contained tetracycline (DIFCO Laboratories, Detroit, USA) on plates, for 24 hours at 37 ° C. More than 10,000 independent transformant clones were obtained, indicating from Clarke and Carbón (Clarke and Carbón, Cell 9, 91-99 (1976)) any chromosomal DNA fragment from Coryneba ct erium mel assecol, which was present at least once in this DNA bank with a probability of 99.9%. The transformants were collected individually and cultured for 24 hours at 37 ° C in 25 μl of LB liquid medium, containing 100 μg / ml of ampicillin, in 96-well microtiter plates. All transformants were plated in duplicate on nylon membranes AMERSHAM Hybondó-N AMERSHAM (Little Chalfont, England) and allowed to develop for 24 hours at 37 ° C. The colonies were used on membranes. The master microtiter plates were stored at -80 ° C until use, then each well was calibrated to 50 μl with 25 μl of 80% glycerol. The selection of the DNA bank of Corynebacterium melsesecol to ATCC 17965 for the presence of the sod gene was carried out by DNA / DNA hybridization with a radioactive probe specific for sod, as described by Ausubel et al. (Ausubel et al., In: Current protocols in molecular biology, John Wiley and sons (eds.), New York, USA (1987)). The DNA probe specific for sod was prepared as follows. Taking into account the codon deviation index for highly expressed genes in Coryneba cteri um species (Malumbres et al., Gene 134, 15-24 (1993)), the degenerate oligonucleotides Si and S2 were designed based on the sequences of peptide 15 and peptide 18 respectively. An additional oligonucleotide, namely S3 was designed from the peptide DMWEHAFYL. These oligonucleotides had the following sequences: 51 5 'GGCCACACCAACCACTCCGTSTT 3' 52 5 'GAGTTGAAGTGRTCCTGGAACTT 3' 53 5 'AGGTAGAAWGCGTGCTCCCACAT 3' (single letter code: S = C or G, R = A or G, W = A or T) The oligonucleotides were synthesized with a Polygen DNA synthesizer from POLYGEN GmbH (Langen, Germany). These oligonucleotides were used to amplify a DNA fragment in a polymerase chain reaction experiment, with the chromosomal DNA of Coryn eba c t erium mel asecol to ATCC 17965, prepared as described above, as the template. This experiment was carried out in an incubation system controlled by the Crocodile II microprocessor of APPLIGENE ONCOR (Illkirch, France), with the Ampli Taq Gold polymerase and its PERKIN ELMER buffer (Foster City, USA), as follows: 30 ng Oligonucleotide chromosomal DNA SI 0.5 μM Oligonucleotide S2 or S3 0.5 μM Ampli Taq Gold 2.5 units Shock absorber xlO 5 μl Water up to a final volume of 50 μl The polymerase chain reaction consisted of incubation for 10 minutes at 94 ° C, followed by 35 cycles of amplification (1 minute of denaturation at 94 ° C, 1 minute of annealing at 50 ° C, 1 minute of polymerization at 72 ° C for each cycle). The size of the amplified DNA fragments, as estimated by the agarose gel electrophoresis, was close to what was expected from the nucleotide sequence data of the known sod genes. The amplification with the oligonucleotides SI and S2 gave a fragment of approximately 130 nucleotides, and the amplification with the oligonucleotides SI and S3 a fragment of approximately 290 nucleotides. Both amplified fragments were cloned in the specialized vector pGEM®-T from PROMEGA CORPORATION (Madison, USA) to give the plasmids pMM6 and pMM7. Plasmid pMM6 contained the fragment amplified with SI and S2, pMM7 contained the fragment amplified with SI and S3. The inserted fragments were sequenced by the dideoxynucleotide chain termination method of Sanger et al. (Sanger et al., Proceedings of the National Academy of Science USA 74, 5463-5467 (1977)), with the oligonucleotide SI as the primer. DNA sequencing was performed with a DNA sequencing system Model 373 from APPLIED BIOSYSTEM (Foster City, USA).

The analysis of the DNA sequence confirmed that both fragments corresponded to the fragments of a gene that codes for superoxide dismutase. The origin of the amplified fragment, obtained with SI and S3 as primers was controlled by the Southern experiment (Southern, Journal of Molecular Biology 98, 503-517 (1975)) as follows. Chromosomal DNA from Coryn eba c t erium melassecol to ATCC 17965 and from Escheri chia coli DH5a (Hanahan, Journal of Molecular Bilogy 166, 557-580 (1983)) were restricted with Hind enzymes III, Pst I or Pvu II. The DNA fragments were separated by horizontal agarose gel electrophoresis, transferred onto a membrane of AMERSHAM Hybondó-N of AMERSHAM (Little Chalfont, England). Oligonucleotides SI and S3 were used to amplify, by the polymerase chain reaction as described above with pMM7 as the template, a DNA fragment specific for sod. This fragment was radioactively labeled with a32P-dCTP with a T7 QuickPrimeo kit from PHARMACIA Biotech (Saint Quentin in Yvelines, France). The marking was done as indicated by the provider. The radioactive probe hybridized specifically with the chromosomal DNA of Corynebacterium um mel assecol to ATCC 17965. This labeled DNA probe was also used to select the genomic library of Coryneba ct erium mel a sesecol to ATCC 17965 previously seeded on plates on membranes of nylon. From this selection, six colonies were first selected. The plasmids were isolated from the respective clones and analyzed by restriction analysis and polymerase chain reaction. Plasmid pMM8 was selected for subsequent studies because it contains a 10 kbp recombinant DNA fragment that hybridizes with the radioactive probe derived from pMM7, from which a 290 nucleotide fragment was amplified by the chain reaction of polymerase with oligonucleotides SI and S3 as primers.

Sequencing of the sod gene The sod locus in the pMM8 plasmid was sequenced by the dideoxynucleotide chain termination method of Sanger et al. (Sanger et al., Proceedings of the National Academy of Science USA 74, 5463-5467 (1977)) on a model 373 DNA sequencing system from APPLIED BIOSYSTEM (Foster City, USA). Sequencing started from the oligonucleotide SI as a primer in one direction, and from the oligonucleotide S3 in the other direction. The determination of the sequence proceeded to the termination on the two strands by means of a "DNA walk" strategy, the new oligonucleotides being designated from the previously determined nucleotide sequences and synthesized as described above. The nucleotide sequence was analyzed by computer using the Gene Jockey II program, Sequence Processor, provided by BIOSOFT (Cambridge, United Kingdom). The sequence is shown in SEQ ID NO. 1. An open reading structure greater than 603 nucleotides was thus identified. This open reading structure corresponds to a protein of 200 amino acids (SEQ ID No. 2). This deduced protein has a calculated molecular weight of 22,103 da, close to the experimental value determined for the superoxide dismutase protein by SDS-PAGE electrophoresis (see above).

The experimentally determined sequences of peptide 15 and peptide 18 are within the deduced protein sequence. In addition, the deduced protein shows extensive identity to other completely sequenced superoxide dismutases, as observed from the comparisons with the sequences in the database of the National Center for Biotechnology Information (http: // www. //www.ncbi.nlm.nih.gov). The superoxide dismutase of Corynebacterium melassecola ATCC 17965 shows identities with those of Nocardia asteroides (65% identity, accession number U02341, Mycobacterium fortuitum (63% identity, accession number X70914), Mycobacterium avium (63% identity, number access U11550), Mycobacterium leprae (61% identity, accession number X16453) and Mycobacterium tuberculosus (60% identity, accession number X52861).

Example 2 Expression of the sod gene 1. Cloning of the sod gene into an expression vector For the expression of the sod gene, the expression vector pMM23 was constructed starting from pCGL482. The plasmid pCGL482 (Figure 2) is a shuttle vector of Es cheri chi a coli -Coryn eba ct eri um gl utami cum composed of segments of pACYC184 (Chang and Cohen, Journal of Bacteriology 134 (3), 1141-1156 (1978) ), pBLl (Fernandez-Gonzales et al., Journal of Bacteriology 176 (11), 3154-3161 (1994)), the origin of replication of phage M13 (Rashed and Oberer, Microbiological Review 50, 401-427, (1986)). )), a multiple cloning site and the chloramphenicol resistance gene of pACYC184 as a selection marker. The DNA of pCGL482 was digested with the restriction enzymes BamHI and Stul. Plasmid pMM8 was digested with Ncol and Smal and the 0.8 kbp DNA fragment comprising the sod gene was isolated by agarose gel electrophoresis. Plasmid pKK388-l (Brosius, in Vector: A survey of molecular cloning vectors and their uses, Rodrigez Editor, pp 205-225, Butterworth Boston, USA (1988)) was digested with BamHI and Ncol and the DNA fragment was 0.36. kpb spanning the tre promoter (Brosius et al., Journal of Biological Chemistry 260, 3539-3541 (1985)) was isolated by agarose gel electrophoresis. The three components were mixed and treated with the T4 DNA ligase. The ligation mixture was used to transform Es cheri chi a coli strain XLl-Blue (Bullock et al, BioTechniques 5, 376-378 (1987))) by electroporation as described by Bonamy et al. (Bonamy et al., FEMS Microbiology Letters 66, 263-270 (1990)). Transformants were selected by incubation on Luria Bertani agar from Bacto® (DIFCO Laboratories, Detroit, USA) supplemented with chloramphenicol (30 μg / ml) for 2 days at 37 ° C. The plasmid DNA was isolated from a single colony and was designated pMM23 (Figure 1). C. mel to ss ecological to strain 1Ó19 (deposited under DSM 12859) which is a restriction deficient mutant, derived from strain ATCC 17965 was transformed with the plasmids pCGL482 and pMM23 as described by Bonamy et al. (Bonamy et al., FEMS Microbiology Letters 66, 263-270 (1990)). Transformants were selected on Bacto® Brain-Heart Infusion Agar (DIFCO Laboratories, Detroit, USA) supplemented with chloramphenicol (6 μg / ml) incubated at 34 ° C for 2 days.

Enzymatic measurements Strains 1019, 1019 / pCGL482 and 1019 / pMM23 were developed aerobically on Bacto® Brain Heart Infusion Medium (DIFCO Laboratories, Detroit, USA) supplemented with chloramphenicol for the last two strains, at 34 ° C, up to the intermediate exponential phase . Strains were harvested by centrifugation using a Megafuge 1.0 R centrifuge from HERAUS Instruments GmbH (Hanau, Germany) at 5,000 xg for 15 minutes and resuspended in 50 mM sodium phosphate buffer pH 8.0 to give a final optical density of '50, as measured by a DU 7400 photometer from BECKMAN (Palo Alto, USA) at a wavelength of 570 nm. The cells were mechanically disintegrated with glass spheres. A volume of 1 ml cell suspension was mixed with 1 g of glass spheres having a diameter of 0.5 mm in a 2 ml Eppendorf tube. The suspension was vortexed for 2 minutes and then incubated on ice for 2 minutes. This procedure was repeated three times. The resulting suspension was centrifuged for at least 15 minutes at 13,000 rpm using a Sigma 113 centrifuge from SIGMA (Saint Louis, USA). The supernatant was used for enzymatic assays. The superoxide dismutase assays were performed as described by Ukeda et al.

(Analytical Biochemistry 251 (2), 206-209 (1997)) using the Mn-dependent superoxide dismutase from Escheri chi a coli obtained from SIGMA-ALDRICH (Saint Quentin Fallavier, France) as a standard. Estimates of protein concentration were made by the Lowry method "(Lowry et al., Journal of Biological Chemistry 193, 265-275 (1951)) using Immunoglobulin G 'Bovine as a standard.The results are shown in the Table 1. An enzyme unit is defined as the amount of enzyme that inhibits the rate of reduction of cytochrome C by 50% in a coupled system, with xanthine and xanthine oxidase at pH 7.8 and 25 ° C.

Table 1 Example 3 Construction of a lysine producer with increased superoxide dismutase Corynebact eri um gl u tami cum strain MH20-22B is a lysine producer known in the art and, for example described in European Patent EP-B-0435132. The strain MH20-22B is deposited in the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSM, Braunschweig, Germany) as DSM5715 according to the Budapest Treaty. Strain MH20-22B fμe transformed with the plasmids pMM23 and pCGL482 as described in Example 2. In this way, the strain was obtained MH20-22B / pMM23 having an increased activity of superoxide dismutase and strain MH20-22B / pCGL482.

Example 4 Production of lysine under oxidative stress Strains MH20-22B and MH20-22B / pMM23 were preculivated in 50 ml of CGIII (Menkel et al., 1989, Applied and Environmental Microbiology 55: 684-688). A 500 ml shake flask was used, protected and the culture was carried out for 19 hours at an initial pH of 7.0, at 33 ° C, and at 250 rpm on a shaker with a rotation diameter of 50 mm. Cells were used to inoculate the main culture with an optical density at 660 nm of 0.3 (Biochrom 'Novaspec 4049, LKB Instrument GmbH, Grafelfing, Germany, cuvette width 10 mm). The main culture was performed in 50 ml of CGC (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) which was modified by the addition of 5 g / 1 of corn syrup liquor, 30 g / 1 of dextrose, 42 g / 1 of morpholinopropanesulfonic acid, and 150 mg / l of leucine, and supplemented with 200 mg / liter of -paraquate. A 500 ml, protected flask was used, and the cultivation was carried out for 20 hours at an initial pH of 7.0, at 33 ° C, and at 250 rpm on a shaker with a rotation diameter of 50 mm. At the end of the fermentation the concentration of the lysine was determined (amino acid analyzer Eppendorf-BioTronik, Hamburg, Germany). The lysine concentrations were 4.8 g / liter in the culture of strain MH20-22B and 6.6 g / liter in the culture of the strain MH20-22B / pMM23. The concentrations are given as lysine hydrochloride.

Example 5 Resistance against redox stress caused by hydrogen peroxide or paraquate The resistance test or the survival test, respectively, was carried out in 10 ml test tubes which were sterilized and filled with 4 ml of sterile GCG-TEST medium described in Table 2. Hydrogen peroxide or paraquate , respectively, were added to the CGC-TEST medium in concentrations as indicated in Table 3 and Table 4. Strains MH20-22B / pMM23 and MH20-22B / pCGL482 were precultivated as described in Example 4. Biomass of the main culture was removed from the culture broth by centrifugation at 15000 g for 10 minutes and used for the subsequent analysis in the survival assay. The concentrate or button was resuspended in CGC-TEST medium to reach an optical density at 660 nm of 1.0. An aliquot of 0.1 ml was transferred to 4 ml of CGC-TEST. The incubation in the 10 ml test tubes was carried out for 20 hours at an initial pH of 7.0, at 37 ° C, and at 250 rpm on a shaker with a rotation diameter of 25 mm. Colony forming units were determined for 20-hour samples by scattered plate placement of 100 μl aliquots at appropriate dilutions on heart-meat infusion agar. The result with the redox tension agent, hydrogen peroxide, which can be used as an oxygen source for the optimization of bacterial growth in the fermentations, is shown in Table 3. The result with the redox tension agent paraquat that promotes the formation of superoxide radicals, shown in Table 4.

Table 2 Concentration component Dextrose (separately sterilized) 5 g / L (NH4) 2S04 5 g / L Urea 5 g / L Biotin (sterilized by filtration) 200 μg / L KH2P04 500 mg / L K2HP04 500 mg / L MgSO4 »7 H20 250 mg / L CaCl2 «2 H20 10 mg / L FeS04» 7 H20 10 mg / L MnSO4 «H20 10 mg / L CuSO4 200 μg / L ZnS04 «7H20 1 mg / L NiCl2« 6H20 20 μg / L The medium was prepared with water from the tap Table 3 MH20-22B / pCGL482 MH20-22B / pMM23 CFU peroxide CFU Reí. hydrogen (g / L (cell / ml) (cell / ml) 0 11.0 106 1.0 4.1 106 1.0 0. 03 3.1 106 0.3 8.9 106 2.2 3. 00 5.0 106 0.5 10.0 106 2.4 CFU: Colony forming units (ml x) Reí: Relative units Table 4 MH20-22B / pCGL482 MH20-22B / pMM23 Paraquate (mg / L) CFU Reí. CFU Reí. (cell / ml) (cell / ml) 0 8.4 106 1.0 4.9 106 1.0 200 4.6 106 0.5 14.0 106 2.9 CFU: Colony forming units (ml a) Reí: Relative units LIST OF SEQUENCES < 110 > Degussa-Hüls AG Center National de La Recherche Scientifique (CNRS) < 120 > A process for the fermentative preparation of metabolic prod and for the nucleotide sequences coding for the sod gene. < 130 > 990078 BT < 140 > < 141 > < 160 > 2 < 1"70> Patentlr Ver. 2.1 <210> <211> 1143 <212> DNA <213> Corynebacterium melassecola ATCC 17965 <220> <221> CDS < 211 > 222 > I338) .. 93 ~ p < 400 > 1 ggatccggcc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag 60 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt 120 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 180 catccggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacagc 240 gccgctgaga aaaagcgaag cggcactgct ctttaacaat ttatcagaca atctgtgtgg 300 gcactcgacc ggaattatcg ataaggaggt ttaaacc atg gct gta tac gaa etc 355 Met Wing Val Tyr Glu Leu 1 5 cea gaa etc gac tac gca tac gac gct etc gag cea cae ate gcc gct 403 Pro Glu Leu Asp Tyr Ala Tyr Asp Ala Leu Glu Pro His lie Wing Wing 1C 15 20 gaa ate atg gag ctt falls fall tec aag falls falls gca acc tac gtt gca 451 Glu lie Met Glu Leu His His Ser Lys His His Wing Thr Tyr Val Wing 25 30 ggc gca gc aac gca gca etc gag gc a cta gag aag gca cgc gaa gag ggc 499 Gly Wing Asn Wing Wing Leu Glu Wing Leu Glu Lys Wing Arg Glu Glu Gly 40 45 50 acc aac ect gac cag ate cgt gca ctg tec aag aac ctt gca ttc aac 547 Thr Asn Pro Aso Gin lie Arg Ala Leu Ser Lys Asn Leu Ala Phe Asn 55 60 65 70 etc ggt gga falls acc aac falls tec gtt ttc tgg aag aac etc tec ect 595 Leu Gly .Gly His Thr Asn His Ser Val Phe Trp Lys Asn Leu Ser Pro 75 80 85 aac ggt ggc ggc gag ect acc ggc gaa ctg gct gag gct ate aac cgc 643 Asp Glv Gly Glu Pro Thr Gly Glu Leu Wing Glu Wing He Asn Arg 5C 95 100 gac ttc ggt tet ttc gct aag ttc cag gat falls ttc aac tet gca gca gca 691 Aso Phe Gly Ser Phe Ala Lys Phe Gln Asp His Phe Asn Ser Wing Ala 105 110 115 etc ggc ctg cag ggc tec ggc tgg gcg gtt etc ggc tac gac falls tet 739 Leu Glv Leu Gin Gly Ser Gly Trp Wing Val Leu Gly Tyr Asp His He 120 125 130 tec ggt cgc etc gtt ate gag cag etc acc gac cag cag ggc aac ate 787 Ser Glv Arg Leu Val He Glu Gln Leu Thr Asp Gln Gln Gly Asn He 13 £ 140 145 150 tec gtc gac ate action gtt ctg atg etc gat atg tgg gag drops gct 835 Ser Val Asp He Thr Pro Val Leu Met Leu Asp Met Trp Glu His Wing 155 160 165 ttc tac cg tg cag tac aag aac gtt aag gca gat tac gtc aag gct gtt 883 Phe Tyr Leu Glr. Tyr Lys Asn Val Lys Wing Asp Tyr Val Lys Wing Val ': ~; 175 180 tgg aac gtc ttc aac tgg gac gac gca gca gca cgc ttc gca gca gct 931 Trp Asn Val Pne Asn Trp Asp Asp Ala Ala Ala Arg Phe Ala Ala Ala 135 190 195 tec aag taagcatttt tagtacgtgc aataaccact ctggtttttc cagggtggtt 987 Ser Lys ttttgatgcc ctt-ttggag tcttcaactg ggtagcgtta ggatteacca tttccggcgg 1047 gcatccggcg aaaaatggtg aatccacaca ctgttgccgg gcagtaagta cttttcgccg 1107 accccgatcs ctasgccagc tgtggcaagc cccggg-- 1143 < 210 > 2 < 21I > 200 < 212 > PRT < 213 > Coryneba-terium melassecola ATCC 17965 < 400 > 2 Met Wing Val Tyr Giu Leu Pro Glu Leu Asp Tyr Wing Tyr Asp Wing Leu 1 5 10 15 Glu Pro His He Wing Wing Glu He Met Glu Leu His His Ser Lys His • 2 25 30 Hxs Wing Thr Tyr Vai Aia Gly Wing Asn Wing Ala Leu Glu Wing Leu Glu 35 40 45 Lys Wing Arg Glu Glu Gly Thr- Asn Pro Asp Gln He Arg Wing Leu Ser 50 55 60 Lys Asn Leu Wing Phe Asn Leu Gly Gly His Thr Asn His Ser Val Phe 65. 70 75 80 Trp Lys Asn Leu Ser Pro Asn Gly Gly Gly Pro Thr Gly Glu Leu 85 90 95 Wing Glu Wing He Asn Arg Asp Phe Gly Ser Phe Wing Lys Phe Gln Asp 100 105 110 His Phe Asn Ser Ala Ala Leu Gly Leu Gln Gly Ser Gly Trp Wing Val 115 120 1? 2 «5 Leu Gly Tyr Asp His He Ser Gly Arg Leu Val He Glu Gln Leu Thr -130 135 140 10 Aso Gln Gln Gly Asn He Ser Val Asp He Thr Pro Val Leu Met Leu 145 150 115555 160 Asp Met Trp Glu His Wing Phe Tyr Leu Gln Tyr Lys Asn Val Lys Wing 165 170 175 15 Asp Tyr Val Lys Wing Val Trp Asn Val Phe Asn Trp Asp Asp Wing Wing 180 185 190 Wing Arg Phe Wing Wing Wing Ser Lys 20 200 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (23)

CLAIMS Having described the invention as above, property is claimed as contained in the following:

1. A preferably recombinant DNA, derived from Coryn eba ct erium, characterized in that it is replicable in coryneform microorganisms and that it contains at least the nucleotide sequence coding for the sod gene represented in SEQ ID NO. 1.

2. The replicable DNA according to claim 1, characterized in that it comprises: (i) the nucleotide sequence, shown in SEQ ID NO. 1, or (ii) at least one sequence corresponding to the sequence (i) within the region of degeneracy of the genetic code, or (iii) at least one sequence that hybridizes with the sequence that is complementary to the sequence (i) ) or (ii), and optionally (iv) sense-neutral, functional mutations in (i).

3. An amino acid sequence for the protein derived from the nucleotide sequences according to claims 1 or 2, represented in SEQ ID NO. 2.

4. The coryneform microorganisms, in particular the genus Coryn eba c t eri um, are characterized because they are transformed by the introduction of one or more of the replicable DNAs according to one of claims 1 or 2.

5. The shuttle vector pMM23, characterized by the restriction diagram given in Figure 1 and deposited in Esch erichi a col i under the name DSM 12860.

6. A process for increasing the activity of superoxide dismutase in coryneform bacteria, characterized in that the sod gene, or the nucleotide sequences encoding it, is amplified, in particular overexpressed.

7. A process for the preparation of metabolic products, in particular L-lysma, by fermentation of coryneform bacteria, characterized in that bacteria are used in which the sod gene or the nucleotide sequences coding for it, in particular overexpressed, are amplified.

8. A process according to claim 7, characterized in that bacteria are used in which other genes are amplified in the biosysthetic pathway of the desired metabolic product.

9. A process according to claim 7, characterized in that bacteria are used in which the metabolic pathways that reduce the production of the desired metabolic product are at least partially quenched.

10. A process according to claims 7 to 9, characterized in that a strain that has been transformed with a plasmid vector is used, and the plasmid vector possesses the nucleotide sequence coding for the sod gene.

11. A process according to claim 9, characterized in that the bacteria transformed with the plasmid vector pMM23, deposited in Esch eri ch i a ccl i under the number DSM 12860, are used.

12. A process according to one or more of the preceding claims, characterized in that coryneform bacteria are used that produce nucleotides, vitamins and amino acids.

13. A process according to one or more of the preceding claims, characterized in that the cormetrical bacteria used produce L-lysine.

14. A process according to claim 8, for producing nucleotides, characterized in that the purF gene encoding glutamine-PRPP-amidotransferase is simultaneously overexpressed.

15. A process according to claim 8, for preparing the nucleotides, characterized in that the carAB gene encoding the carbamoyl synthetase is simultaneously overexpressed.

16. A process according to claim 8, for preparing D-pantothenic acid, characterized in that the panD gene coding for aspart ato-decarboxylase is simultaneously overexpressed.

17. A process according to claim 8 for preparing L-lysine, characterized in that the dapA gene coding for dihydrohipicolinate synthase is simultaneously overexpressed.

18. A process of claim 8, for preparing L-lysine, characterized in that a DNA fragment that promotes resistance to S- (2-aminoethyl) cis-theine is simultaneously amplified.

19. A process for the fermentative preparation of the desired metabolic products according to one or more of the preceding claims, characterized in that the following steps are carried out: (a) the fermentation of the coryneform bacteria that produce the desired metabolic product, in which the sod gene is less amplified, b) the enrichment of the desired metabolic product in a medium or in the cells of the bacteria and c) the isolation of the desired product.

20. A process for the fermentative preparation of L-amino acids according to claim 19, characterized in that the following steps are carried out: a) the fermentation of the coryneform bacteria that produce the desired metabolic product, in which at least the sod gene is amplified , b) the enrichment of the desired metabolic product in a medium or in the cells of the bacteria and c) the isolation of the desired product.

21. A process for the fermantative preparation of D-pantothenic acid according to claim 19, characterized in that the following steps are carried out: a) the fermentation of the coryneform bacteria that produce D-pantothenic acid, in which at least the gen sod, b) the enrichment of D-pantothenic acid in a medium or in the cells of the bacterial and c) the isolation of this acid.

22. A process according to any of claims 19, 20 or 21, optionally in the presence of an oxygen-containing compound.

23. A process according to claim 22, characterized in that the oxygen-containing compound is C € hydrogen peroxide.